1. Field of the Invention
The present invention relates to a plant-infectious nucleic acid molecule from Pepper mottle virus, and a viral vector a transformed cell and a transgenic plant having it.
2. Background of Technique
Various aspects of virus pathogenicity have been studied using in vitro- or in vivo-transcribed infectious RNA derived from full-length cDNA clones. It is not known how virus proteins are expressed from full-length clones, since the vector sequences do not contain promoters expected to transcribe the virus RNA in bacterial cells. Fakhfakh and their colleagues suggested that viral RNA is transcribed from cryptic promoters and protein synthesis initiated at cryptic ribosomal binding sites present in the virus cDNA sequences (Fakhfakh et al., 1996).
However, manipulation and amplification of full-length clones may prove difficult due to instability or toxicity of some virus sequences in bacteria (Chen and Hruening, 1992; Boyer and Haenni, 1994; Fakhfakh et al., 1996). Expression of virus proteins in E. coli has been reported to have toxic effects on the host cells (Lama and Carrasco, 1992; Rodriguez and Shaw, 1991). The toxic effects from undesired protein expression can be relieved by cloning in E. coli strains that reduce the plasmid copy number (Greener, 1993) or using low copy number cloning vectors (Schweizer, 2008).
Among potyviruses, the in vitro synthesis of biologically active RNAs from full-length cDNA clone with bacterial phage promoters have been reported; Tobacco vein mottling virus (TVMV) (Domier et al., 1989; Nicolas et al., 1996), Plum pox virus (PPV) (Riechmann et al., 1990), Zucchini yellow mosaic virus (ZYMV) (Gal-On et al., 1991; Lin et al., 2002), Tobacco etch virus (TEV) (Dolja et al., 1992), Peanut stripe virus (PStV) (Flasinski et al., 1995), Pea seed-borne mosaic virus (PSbMV) (Johansen et al, 1996), Potato virus A (PVA) (Puurand et al., 1996), Papaya ringspot virus (PRSV) (Chiang and Yeh, 1997), Potato virus Y (PVY) (Jakab et al., 1997), Papaya ringspot virus (PRSV) (Chiang and Yeh, 1997), Turnip mosaic virus (TuMV) (Sanchez et al, 1998) and Johnsongrass mosaic virus (JGMV-Jg)(Kim et al., 2003) and so on.
Another system is based on the delivery of particles coated with cDNA or the plasmids directly introduced of a virus into the plant cell to induce infection. In vivo infectious transcripts, which are driven by a Cauliflower mosaic virus (CaMV) 35S promoter that can be transcribed by an endogenous host RNA polymerase, have been reported for PPV-NAT (Maiss et al., 1992), ZYMV (Gal-On et al., 1995), PVY-NTN (Fakhfakh et al., 1996), PSbMV (Johansen, 1996), Clover yellow vein virus (CIYVV) (Takahashi et al, 1997), PRSV (Chiang and Yeh, 1997), PVY-N605 (Jakab et al, 1997), TuMV (Sanchez et al., 1998), Lettuce mosaic virus (LMV) (Yang et al., 1998) and PSbMV-L1 (Olsen and Johansen, 2001). In vitro- or in vivo-transcribed infectious RNA derived from full-length cDNA clones are an important tool in the study of RNA viruses. These clones are possible to facilitated studies of non-destructive monitoring of virus infection without by tagging reporter genes, such as green fluorescence protein gene (GFP) or β-glucuronidase gene (GUS).
Among the potyviruses, TEV was first developed to express reporter gene (Dolja et al., 1992; Carrington et al., 1993). Later, many potyviruses such as PPV (Guo et al., 1998; Fernandez-Fernandez et al., 2001), LMV (German-Retana et al., 2000), CIYVV (Masuta et al., 2000), Wheat streak mosaic virus (WSMV) (Choi et al., 2000), Tobacco vein mottling virus vector (TVMV) (Dietrich and Maiss, 2003), ZYMV (Arazi et al., 2001; Hsu et al., 2004), PVA (Ivanov et al., 2003) and TuMV (Beauchemin et al., 2005) have been engineered into effective expression of reporter gene at different insertion site of virus genome.
Potyviral proteins are expressed by proteolytic processing of the large precursor polyprotein by three virus-encoded proteases, P1, HC-pro and NIa. P1 and HC-pro automatically cleave at their respective C termini, and NIa cleave the remains (Uyeda, 1997). Most of the foreign ORFs are constructed adjacent to the junction between P1 and HC-Pro or NIb and CP by directional insertion. A plant virus-based vector is a useful tool for efficient expression of target foreign proteins in plants. Plant expression systems have a significant advantage compared to other methods of recombinant protein production since plants are much cheaper and easier in cultivation than cell cultures. This system provides rapid and transient expression of heterogonous genes systemically in plants. These virus-based vectors have been used to express genes of pharmaceutical, agronomic value, elicit genetically dominant, gene-silencing phenotypes in plants to determine the functions of unknown genes (Donson et al., 1991; Kumagai et al., 1993, 1995; Masuta et al., 2000; Arazi et al., 2001; Fitzmaurice et at., 2002). They have also been used to produce proteins applicable to various therapeutic interventions and vaccine components that are applicable as therapeutic cancer vaccines (McCormick et al., 1999) Further, expression of sequences in plants by virus expression vectors can result in reprogramming specific metabolic pathways in plants through virus-induced gene-silencing (VIGS) effects (Baulcombe et al., 1999) or protein expression (Fitzmaurice et al., 2002). Heterologous expression of a cDNA for capsanthin-capsorubin synthase (ccs) in N. benthamiana resulted in an orange-red phenotype and the accumulation of novel carotenoids capsanthin and capsorubin (Kumagai et al., 1998).
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.